Acta physiol. scand. 1976. 96. 256-266 From The Institute of Physiology, University of Bergen, Norway
Regional Distribution of Blood Flow in Calf Muscles of Rat during Passive Stretch and Sustained Contraction BY A. WISNESand A.
KIRKEBB
Received 4 August 1975
Abstract WISNES, A. and A. KIRKEBB. Regional distribution of blood.flow in carf muscles of’ rat durinR passive stretch and sustained contraction. Acta physiol. scand. 1976. 96. 256-266. The regional distribution and number of microspheres in the calf muscles of rat has been studied during isometric sustained contraction and in stretched uncontracted muscles in situ. Carbonized microspheres, I 5 + 5 Icm, were injected into the aortic arch and muscle blood flow arrested 6 sec later. The calf muscles were freeze sectioned (12 slices of 40 pn) and the microspheres counted microscopically. The microsphere concentration in the gastrocnemius and plantaris muscles during rest was 4.6k 1.6 spheres/mm3 (mean S.E.). One min after a standardized exercise programme the sphere concentration was increased to 20.5 3.9 spheres/mm3. At increasing force of contraction following the standard exercise programme, the microsphere concentration fell from 1 l . 6 f 2 . 5 at 2 5 % of maximal force of contraction (MFC) to 2.2+ 0.6 spheres/mm3 at 100% MFC (5.6 kg/cm2). Corresponding measurements in stretched, uncontracted muscles showed a similar fall in microsphere concentration when stretch was increased from 50 to 175% MFC. The ratio between microsphere concentration in the central inner zone and in the peripheral outer zone was slightly higher than unity (1.08-1.16) in muscles at rest and at light postexercise hyperemia. At 75 and 100% MFC the ratio was 0.76+0.07 and 0.57k0.13, significantly lower than unity. Stretching of the uncontracted muscle group to 175% MFC reduced the ratio towards zero. The greater reduction in blood flow to the inner central zone of contracted calf muscles shown by microsphere distrihution was confirmed by measurement of lZ61-antipyrinedistribution. These results show an increased resistance against blood flow during active contraction or stretching of the calf muscles, most pronounced in the central inner zone at high tensions.
Gaskell (1877) showed that muscular blood flow decreased during contraction of dog calf muscles, a finding that has been confirmed in many later works. Gray and Staub (1967) observed less reduction of blood flow during passive stretching than by the same degree of active tension. Hirche, Raff and Grun (1970), working on papaverine dilated muscles, found an equal increase in resistance by passive or active tension. In spindle shaped muscles the transverse component of force of contracting fibers may effect a hydrostatic or fiber pressure on vessels that is higher in regions close to the central axis than in the periphery of the muscles. An effect of this kind, for instance in calf muscles, could cause a lower blood flow to central than to peripheral regions during contraction.
DISTRIBUTION OF CALF MUSCLE BLOOD FLOW
257
The effect might be most pronounced in contracted muscle having a more spherical shape than muscle passively stretched. Local reduction of blood flow may possibly be the cause for ischemic pain in tense muscles. The purpose of the present study was to investigate blood flow distribution in contracted and in passively stretched spindle formed skeletal muscles, by means of microsphere and Y-antipyrine techniques. These methods have been used for measurements of regional distribution of blood flow in the heart (Yipintsoi et a/. 1971, Domenech et al. 1969), but not in skeletal muscle. We have found the methods suitable for measurements of flow distribution to small areas within one muscle, provided that the microsphere counts are high enough to give the statistical precision needed.
Methods The study was performed on 64 male Wistar rats (mean weight 440 g), anesthetized with sodium pentobarbital (40 mg/kg b.wt.) injected intraperitoneally. The right hindleg of the rat was fixed t o a knee holder by a drill through the femur condyles. Supporting bars kept the pubic and tibia1 bones in a fixed position. Care was taken to keep the calf muscles in their natural position and to remove as little as possible of skin and fascia. The tuber calcaneum with Achilles tendon and the plantaris tendon were cut and sutured to a steel wire connected to a strain gauge. Shielded bipolar electrodes were attached on the tibialis nerve 6-7 mm apart to secure homogeneous stimulation across the nerve. Different forces of contraction were obtained by varying the voltage of stimulation (0-7 V) at fixed pulse frequency (50 Hz) and duration (0.15 ms). The calf muscles were stretched passively at 100 g and the maximum isometric force of contraction (MFC) recorded on a Brush recorder. To attain a moderate vasodilation and thereby higher microsphere counts the muscle was contracted tetanically 6 times at 50% MFC, each time for 3 s. During a 1 min pause a sonified solution of 7 x 10Bcarbonizedmicrospheres (Nuclear Products, 3 M Co.) with diameter 15+5 p m in 350 pl Rheomacrodex was introduced into a calibrated tube. This tube was then connected to a catheter (PE 50) with its tip located at the origin of the left carotid artery. After a short test contraction the tetanic contraction o r stretch was started and the microsphere or lzKl-antipyrine solution injected within 1.5 s. 6 s later blood flow to the calf muscles was arrested by pulling a wire sling around the thigh. The rat was killed before the pull was released. Sustained isometric contraction, microsphere technique
-
The first series of experiments (n 26) was divided in 6 separate groups of sustained isometric contraction: 0‘yo6,25nTo,,50”:. 75%, IOO”, MFC, starting at light postexercise hyperemia and an extra resting control group (rest) not exercised before o r during injection. The calf muscles were freeze sectioned transversely to the muscle axis. 12 sections, each 40 p m thick, were sampled in series i n the region of the greatest diameter of the muscle group. The microspheres were counted with microscope and incident light at 36 times magnification. At this magnification it was impossible to view the whole section. A chequered piece of film with a standardized muscle outline reference and a central cross was oriented on the tissue section t o be examined. The outline reference area and thereby the muscles were divided in an inner central zone (I) (0.5 cmz) and an outer peripheral zone (0) (0.75-0.80 cmz). By a drawing mirror the microspheres could be plotted on a paper copy of the film, allowing systematic counting of the microspheres situated in the 2 zones. The microspheres in the soleus muscle were counted separately. Passice stretching, microsphere technique
In the second series of experiments (n -22) on the effect of passive stretching of uncontracted muscle, the muscles were prepared and stimulated, as described above, before the injection of microspheres. The force of stretching was regulated to 0%. 500/0, loo%, 125%, 150% and 175% MFC by a screw shortening the wire between the tendon and the strain gauge. The first and second series had control groups ( n = 8 ) in common. I7 - 76.5872
258
A. WISNES AND A. KIRKEBB
Susrained isometric contraction, la61-antipyrine technique In the third series of experiments (n=24) on 5 groups of 0%. 2 5 % , 75% and 100% MFC of sustained, isometric contraction and of rest, the experimental procedure was identical to the procedure in the first series except for the following steps. At start of the contraction 10 pCi 1261-antipyrine(NEN Chemical GmbH) in 100 pl saline was injected within 1 s into aorta. 6 s later the calf muscle group was rapidly removed and frozen in isopentan cooled to 160°C. Disks (h 2-4 mm) of muscle were cut into inner (0.5 a n a ) and outer (0.754.80 cm2)zone samples by a knife shaped as the standard inner zone outline described above. Radioactivities in 0.24.5 g weighed muscle pieces were counted in a welltype scintillation counter. The soleus muscle was included in the outer zone. Using Students unpaired t-test, differences were considered significant at p < 0.05.
+
Results Microsphere method
By sampling through a catheter placed distally in the femoral artery it was found that the highest concentration of microspheres in the blood stream passed the femoral artery about 2 s after start of injection into the aortic arch. Within 6 s at least 75% of the microspheres to the leg had passed into the sampling catheter. Contraction and simultaneous injection of a standard dose of microspheres caused the arterial pressure to rise briefly by 20-30 mm Hg, mainly due to the effect of the injected volume of fluid. Contraction without injection gave a minimal rise in arterial pressure. The number of microspheres in the 12 sections of each muscle showed some scatter. By a x* test it was shown that in only 4 out of 26 muscles the numbers of spheres per section were distributed significantly (p
Regional Distribution of Blood Flow in Calf Muscles of Rat during Passive Stretch and Sustained Contraction BY A. WISNESand A.
KIRKEBB
Received 4 August 1975
Abstract WISNES, A. and A. KIRKEBB. Regional distribution of blood.flow in carf muscles of’ rat durinR passive stretch and sustained contraction. Acta physiol. scand. 1976. 96. 256-266. The regional distribution and number of microspheres in the calf muscles of rat has been studied during isometric sustained contraction and in stretched uncontracted muscles in situ. Carbonized microspheres, I 5 + 5 Icm, were injected into the aortic arch and muscle blood flow arrested 6 sec later. The calf muscles were freeze sectioned (12 slices of 40 pn) and the microspheres counted microscopically. The microsphere concentration in the gastrocnemius and plantaris muscles during rest was 4.6k 1.6 spheres/mm3 (mean S.E.). One min after a standardized exercise programme the sphere concentration was increased to 20.5 3.9 spheres/mm3. At increasing force of contraction following the standard exercise programme, the microsphere concentration fell from 1 l . 6 f 2 . 5 at 2 5 % of maximal force of contraction (MFC) to 2.2+ 0.6 spheres/mm3 at 100% MFC (5.6 kg/cm2). Corresponding measurements in stretched, uncontracted muscles showed a similar fall in microsphere concentration when stretch was increased from 50 to 175% MFC. The ratio between microsphere concentration in the central inner zone and in the peripheral outer zone was slightly higher than unity (1.08-1.16) in muscles at rest and at light postexercise hyperemia. At 75 and 100% MFC the ratio was 0.76+0.07 and 0.57k0.13, significantly lower than unity. Stretching of the uncontracted muscle group to 175% MFC reduced the ratio towards zero. The greater reduction in blood flow to the inner central zone of contracted calf muscles shown by microsphere distrihution was confirmed by measurement of lZ61-antipyrinedistribution. These results show an increased resistance against blood flow during active contraction or stretching of the calf muscles, most pronounced in the central inner zone at high tensions.
Gaskell (1877) showed that muscular blood flow decreased during contraction of dog calf muscles, a finding that has been confirmed in many later works. Gray and Staub (1967) observed less reduction of blood flow during passive stretching than by the same degree of active tension. Hirche, Raff and Grun (1970), working on papaverine dilated muscles, found an equal increase in resistance by passive or active tension. In spindle shaped muscles the transverse component of force of contracting fibers may effect a hydrostatic or fiber pressure on vessels that is higher in regions close to the central axis than in the periphery of the muscles. An effect of this kind, for instance in calf muscles, could cause a lower blood flow to central than to peripheral regions during contraction.
DISTRIBUTION OF CALF MUSCLE BLOOD FLOW
257
The effect might be most pronounced in contracted muscle having a more spherical shape than muscle passively stretched. Local reduction of blood flow may possibly be the cause for ischemic pain in tense muscles. The purpose of the present study was to investigate blood flow distribution in contracted and in passively stretched spindle formed skeletal muscles, by means of microsphere and Y-antipyrine techniques. These methods have been used for measurements of regional distribution of blood flow in the heart (Yipintsoi et a/. 1971, Domenech et al. 1969), but not in skeletal muscle. We have found the methods suitable for measurements of flow distribution to small areas within one muscle, provided that the microsphere counts are high enough to give the statistical precision needed.
Methods The study was performed on 64 male Wistar rats (mean weight 440 g), anesthetized with sodium pentobarbital (40 mg/kg b.wt.) injected intraperitoneally. The right hindleg of the rat was fixed t o a knee holder by a drill through the femur condyles. Supporting bars kept the pubic and tibia1 bones in a fixed position. Care was taken to keep the calf muscles in their natural position and to remove as little as possible of skin and fascia. The tuber calcaneum with Achilles tendon and the plantaris tendon were cut and sutured to a steel wire connected to a strain gauge. Shielded bipolar electrodes were attached on the tibialis nerve 6-7 mm apart to secure homogeneous stimulation across the nerve. Different forces of contraction were obtained by varying the voltage of stimulation (0-7 V) at fixed pulse frequency (50 Hz) and duration (0.15 ms). The calf muscles were stretched passively at 100 g and the maximum isometric force of contraction (MFC) recorded on a Brush recorder. To attain a moderate vasodilation and thereby higher microsphere counts the muscle was contracted tetanically 6 times at 50% MFC, each time for 3 s. During a 1 min pause a sonified solution of 7 x 10Bcarbonizedmicrospheres (Nuclear Products, 3 M Co.) with diameter 15+5 p m in 350 pl Rheomacrodex was introduced into a calibrated tube. This tube was then connected to a catheter (PE 50) with its tip located at the origin of the left carotid artery. After a short test contraction the tetanic contraction o r stretch was started and the microsphere or lzKl-antipyrine solution injected within 1.5 s. 6 s later blood flow to the calf muscles was arrested by pulling a wire sling around the thigh. The rat was killed before the pull was released. Sustained isometric contraction, microsphere technique
-
The first series of experiments (n 26) was divided in 6 separate groups of sustained isometric contraction: 0‘yo6,25nTo,,50”:. 75%, IOO”, MFC, starting at light postexercise hyperemia and an extra resting control group (rest) not exercised before o r during injection. The calf muscles were freeze sectioned transversely to the muscle axis. 12 sections, each 40 p m thick, were sampled in series i n the region of the greatest diameter of the muscle group. The microspheres were counted with microscope and incident light at 36 times magnification. At this magnification it was impossible to view the whole section. A chequered piece of film with a standardized muscle outline reference and a central cross was oriented on the tissue section t o be examined. The outline reference area and thereby the muscles were divided in an inner central zone (I) (0.5 cmz) and an outer peripheral zone (0) (0.75-0.80 cmz). By a drawing mirror the microspheres could be plotted on a paper copy of the film, allowing systematic counting of the microspheres situated in the 2 zones. The microspheres in the soleus muscle were counted separately. Passice stretching, microsphere technique
In the second series of experiments (n -22) on the effect of passive stretching of uncontracted muscle, the muscles were prepared and stimulated, as described above, before the injection of microspheres. The force of stretching was regulated to 0%. 500/0, loo%, 125%, 150% and 175% MFC by a screw shortening the wire between the tendon and the strain gauge. The first and second series had control groups ( n = 8 ) in common. I7 - 76.5872
258
A. WISNES AND A. KIRKEBB
Susrained isometric contraction, la61-antipyrine technique In the third series of experiments (n=24) on 5 groups of 0%. 2 5 % , 75% and 100% MFC of sustained, isometric contraction and of rest, the experimental procedure was identical to the procedure in the first series except for the following steps. At start of the contraction 10 pCi 1261-antipyrine(NEN Chemical GmbH) in 100 pl saline was injected within 1 s into aorta. 6 s later the calf muscle group was rapidly removed and frozen in isopentan cooled to 160°C. Disks (h 2-4 mm) of muscle were cut into inner (0.5 a n a ) and outer (0.754.80 cm2)zone samples by a knife shaped as the standard inner zone outline described above. Radioactivities in 0.24.5 g weighed muscle pieces were counted in a welltype scintillation counter. The soleus muscle was included in the outer zone. Using Students unpaired t-test, differences were considered significant at p < 0.05.
+
Results Microsphere method
By sampling through a catheter placed distally in the femoral artery it was found that the highest concentration of microspheres in the blood stream passed the femoral artery about 2 s after start of injection into the aortic arch. Within 6 s at least 75% of the microspheres to the leg had passed into the sampling catheter. Contraction and simultaneous injection of a standard dose of microspheres caused the arterial pressure to rise briefly by 20-30 mm Hg, mainly due to the effect of the injected volume of fluid. Contraction without injection gave a minimal rise in arterial pressure. The number of microspheres in the 12 sections of each muscle showed some scatter. By a x* test it was shown that in only 4 out of 26 muscles the numbers of spheres per section were distributed significantly (p
